RESEARCH ARTICLE
Canonical Wnt Signaling Drives Tumor-Like Lesions from Sox2-Positive Precursors of the Murine Olfactory Epithelium Nils W. Engel1,2, Julia E. Neumann1,3, Julia Ahlfeld1¤a, Annika K. Wefers1¤b, Daniel J. Merk1¤c, Jasmin Ohli1, Ulrich Schu¨ller1,3,4,5* 1 Center for Neuropathology, Ludwig-Maximilians-University, Munich, Germany, 2 Department of Internal Medicine II, University Medical Center, Hamburg-Eppendorf, Germany, 3 Institute of Neuropathology, University Medical Center, Hamburg-Eppendorf, Germany, 4 Research Institute Children’s Cancer Center, Hamburg, Germany, 5 Department of Pediatric Hematology and Oncology, University Medical Center, Hamburg-Eppendorf, Germany
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¤a Current address: Division of Clinical Pharmacology, Department of Internal Medicine IV, LudwigMaximilians-University, Munich, Germany ¤b Current address: Department of Neuropathology, Institute of Pathology, University of Heidelberg, Germany ¤c Current address: Dana-Farber Cancer Institute, Boston, MA, United States of America *
[email protected]
Abstract OPEN ACCESS Citation: Engel NW, Neumann JE, Ahlfeld J, Wefers AK, Merk DJ, Ohli J, et al. (2016) Canonical Wnt Signaling Drives Tumor-Like Lesions from Sox2Positive Precursors of the Murine Olfactory Epithelium. PLoS ONE 11(11): e0166690. doi:10.1371/journal.pone.0166690 Editor: Chunming Liu, University of Kentucky, UNITED STATES Received: August 2, 2016 Accepted: November 2, 2016 Published: November 30, 2016 Copyright: © 2016 Engel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Deutsche Krebshilfe, grant number 111630. Competing Interests: The authors have declared that no competing interests exist.
Canonical Wnt signaling is known to promote proliferation of olfactory stem cells. In order to investigate the effects of a constitutive activation of Wnt signaling in Sox2-positive precursor cells of the olfactory epithelium, we used transgenic mice that allowed an inducible deletion of exon 3 of the Ctnnb1 gene, which is responsible for the phosphorylation and degradation of Ctnnb1 protein. After induction of aberrant Wnt activation by Ctnnb1 deletion at embryonic day 14, such mice developed tumor-like lesions in upper parts of the nasal cavity. We still observed areas of epithelial hyperplasia within the olfactory epithelium following early postnatal Wnt activation, but the olfactory epithelial architecture remained unaffected in most parts when Wnt was activated at postnatal day 21 or later. In summary, our results suggest an age-dependent tumorigenic potential of aberrant Wnt signaling in the olfactory epithelium of mice.
Introduction The mammalian olfactory epithelium (OE) is a sensory neuroepithelium, which is unique for its ability of sustaining robust peripheral neurogenesis throughout the lifetime of mice and man [1–4]. The complex regulatory pathways underlying this physiologic capacity have been a matter of intensive biomedical research, given the hope, that major insights here might sustainably transform the field of regenerative medicine in the future. Only recently, the crucial role of the canonical Wnt signaling pathway in the regulation of OE stem cells and progenitors during development and regeneration has been pointed out [5, 6]. Wang et al. showed, that Wnt signaling promotes self-renewal of Sox2-positive stem
PLOS ONE | DOI:10.1371/journal.pone.0166690 November 30, 2016
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Wnt-Driven Lesions in the Olfactory Epithelium
cells and proximate progenitors of the OE in vitro [5]. Moreover, Wnt signaling was found to promote neurogenesis at the expense of non-neuronal differentiation within the OE. Following OE injury in adult mice, strong Wnt activation seems to be necessary for OE regeneration in vivo. In this context, Chen et al. confirmed that Wnt activation may have a pivotal role in OE progenitor cell proliferation and neurogenesis in vivo, and they further suggested that Wnt signaling delays the terminal differentiation of neuronal progenitors into mature olfactory sensory neurons, therefore promoting the expansion of OE immature neuronal progenitors [6]. Beyond the physiological role of Wnt signaling for the regulation of cellular growth and differentiation throughout the body, pathological activation of the canonical Wnt signaling pathway has been identified to play a crucial role in a wide range of human neoplastic diseases [7– 13]. Of note, the Wnt pathway hereby often takes up important physiologic functions and has devastating tumor-causing potential in cases of dysregulation within the same tissue. This is exemplarily true for intestinal tissue homeostasis [14, 15] and colon cancer formation [16–18]. In the central nervous system, Wnt signaling has for instance crucial roles in hindbrain development [19–22] and may induce WNT medulloblastoma formation [12]. This relation is even generalizable to the well-established link between the essential role of certain signaling pathways for brain development and their contribution to the pathogenesis of various human pediatric brain tumors [23]. Here, we sought to answer the question, whether the above outlined parallelism is valid in an OE related context, which would imply a tumorigenic potential of aberrant Wnt signaling within the OE. Thus, we constructed an inducible Sox2-creERT2::Ctnnb1(ex3)Fl/+ mouse model in order to effectively delete exon 3 of Ctnnb1 in Sox2-positive cells of the mouse OE. The resulting alterations in OE architecture were then analyzed on the level of histomorphology and immunohistochemistry.
Material and methods Transgenic mice Sox2-creERT2 [24] and B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J [25] mice were obtained from the Jackson Laboratory. Ctnnb1(ex3)Fl/FL [26] mice were a kind gift from Dr. M. Taketo (University of Tokyo, Japan). Genotyping was performed by PCR analysis using genomic DNA from ear biopsies. Primers for Cre and Ctnnb1(ex3) have been previously published [26, 27], and primers to detect the tdTomato allele have been designed as recommended by the Jackson Laboratory (www.jax.org). In vivo induction of the tdTomato or the defective Ctnnb1(ex3) allele in Sox2-positive cells was achieved by intraperitoneal tamoxifen (Sigma-Aldrich) injection. Pregnant females were injected once with 1 mg of tamoxifen (50 mg/kg body weight in corn oil (Sigma-Aldrich) at E14.5. The day of vaginal plug detection was considered E0.5. Postnatal mice were induced at days P7, P14 or P21 via single tamoxifen injection, likewise. Injected mice were monitored daily for signs of tumor formation or general failure to thrive. Together with littermates not bearing the Cre allele, mutant animals destined for further histological workup were sacrificed shortly after the onset of symptoms by cervical dislocation. All mouse procedures were performed according to protocols approved by the government of Upper Bavaria (#2532-10-14). All efforts were made to ameliorate suffering of mice. Mice were sacrificed by cervical dislocation as soon as any kind of suffering, deterioration of the general health condition or neurological symptoms were visible. This given, analgesics or anesthetics were not applied in order to reduce suffering. Mice were monitored at least once a day. There were no unexpected deaths within this study.
PLOS ONE | DOI:10.1371/journal.pone.0166690 November 30, 2016
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Tissue collection, fixation and H&E staining Sacrificed animals destined for histological evaluation were immediately dissected for tissue collection. The skull bases were formalin-fixed, decalcified in 10% EDTA (pH7.4) and embedded in paraffin according to standard lab protocols. For each of those animals, brain, heart, lungs, gastrointestinal organs and kidneys were collected and separately fixed and embedded according to standard lab protocols. The preserved skull base specimens were cut in frontal orientation until a level with intersection of ethmoturbinates 1–5 was reached. Sections from this level were collected and H&E stains were acquired according to standard lab protocols. For all other organs, representative H&E stains in suitable orientation were evaluated for histomorphological alterations in mutant organs compared to controls. If alterations were detectable, further immunohistochemical staining was performed as described below.
Immunohistochemistry Sections of paraffin-embedded tissue were deparaffinized and rehydrated before heat-induced antigen retrieval was conducted at 100˚C for 20 min in 10 mM sodium citrate buffer for all antibodies. Immunohistochemical staining was done using the primary antibodies in concentrations as follows: RFP: 1:200, Antibodies Online, Cat N˚ AA234; Sox2: 1:200, Abcam, Cat N˚ AB79351; Ki67: 1:200, Abcam, Cat N˚ AB16667, Beta-Catenin: 1:1000, BD Pharmigen, Cat N˚ 610153; Mash1: 1:25, BD Bioscience, Cat N˚ 556604; Chromogranin A: 1:300, Abcam, Cat N˚ AB15160; CD56: undiluted, Ventana Medical Systems, Roche, Cat N˚ 7602625; S100: 1:2000, DAKO, Cat N˚ Z0311; SMA: 1:1000, DAKO, Cat N˚ M0851. Mash1 antibody was applied in blocking puffer over night at room temperature [28]. All other antibodies were applied over night at 4˚C. The HRP/DAB Staining System (DAKO) was used according to the manufacturer’s specifications. Hemalaun was used for nuclear counterstaining. All histological photomicrographs were taken digitally using an Olympus BX50 microscope in combination with the Color View Soft imaging system. For immunofluorescent staining, sections were washed twice with PBS/0.1% Triton X-100 and then incubated in blocking buffer (I-Block protein- based blocking reagent; Applied Biosystems) for 30 minutes. Primary antibodies (RFP: 1:200, Antibodies Online, Cat N˚ AA234; Sox2: 1:200, Abcam, Cat N˚ AB79351; Tuj1: 1:100, BABCO, Cat N˚ MMS435P) were diluted in blocking buffer and applied over night at 4˚C. Next, sections were washed twice with PBS/0.1% Triton X-100 and incubated for another 60 minutes with a 1:500 dilution of fluorescence-labeled secondary antibodies (goat anti-rabbit Alexa546; goat anti-mouse Alexa488, Invitrogen) in blocking buffer. Sections were washed twice with PBS/ 0.1% Triton X-100, counterstained with 4’,6-diamidino-2-phenylindole (DAPI), and mounted in Fluorescent Mounting Medium (DAKO). Images of tissue sections were collected on a Zeiss LSM 780 laser scanning microscope in combination with the ZEN 2012 imaging software. Whole-mount images of stomachs were collected on a Leica DFC3000 G microscope camera in combination with the Leica Application Suite Software.
Statistical analysis Data of survival, body weight and body seize of mice were analyzed using the Prism5 software (GraphPad). Survival of mice was analyzed using Kaplan–Meier survival curves, and the log rank test was used to examine the significance of results. P-values
